Search

Autochthonous yeasts of oenological origin are adapted to highly stressful and selective environments, which makes them potential candidates for probiotics. The objective of the present study was to explore the probiotic potential of 96 native yeasts of oenological origin, their biosafety, resistance to gastrointestinal tract conditions and adhesion properties. Regarding biosafety, 66 isolates shown negative hemolytic activity, negative urease activity and susceptibility to 3 or more antifungals. After the gastrointestinal resistance test, 15 isolates were selected that showed growth at different temperatures, tolerance to low pH and the presence of bile salts in in vitro tests. In general, survival after simulated conditions of the gastrointestinal tract was high and more restrictive was the duodenal. The results of the adhesion properties showed highly variable hydrophobicity and a high percentage of autoaggregation at 24 h. The maximum production of biofilm was detected in the Pichia strains. Of a total of 96 yeast strains, 15 non-Saccharomyces yeasts presented suitable properties as probiotic candidates. The native winemaking strains performed better than the reference probiotic strain, Saccharomyces cerevisiae var. boulardii CNCM I-745, which reaffirms that these strains are promising probiotic candidates and further studies are necessary to confirm their probiosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Examination of the changes in order and arrangement of homologous genes is key for understanding the mechanisms of genome evolution in eukaryotes. Previous comparisons between eukaryotic genomes have revealed considerable conservation across species that diverged hundreds of millions of years ago (e.g., vertebrates, 1 , 2 , 3 bilaterian animals, 4 , 5 and filamentous fungi 6 ). However, understanding how genome organization evolves within and between eukaryotic major lineages remains underexplored. We analyzed high-quality genomes of 120 representative budding yeast species (subphylum Saccharomycotina) spanning ∼400 million years of eukaryotic evolution to examine how their genome organization evolved and to compare it with the evolution of animal and plant genome organization. 7 We found that the decay of both macrosynteny (the conservation of homologous chromosomes) and microsynteny (the conservation of local gene content and order) was strongly associated with evolutionary divergence across budding yeast major clades. However, although macrosynteny decayed very fast, within ∼100 million years, the microsynteny of many genes-especially genes in metabolic clusters (e.g., in the GAL gene cluster 8 )-was much more deeply conserved both within major clades and across the subphylum. We further found that when genomes with similar evolutionary divergence times were compared, budding yeasts had lower macrosynteny conservation than animals and filamentous fungi but higher conservation than angiosperms. In contrast, budding yeasts had levels of microsynteny conservation on par with mammals, whereas angiosperms exhibited very low conservation. Our results provide new insight into the tempo and mode of the evolution of gene and genome organization across an entire eukaryotic subphylum., Competing Interests: Declaration of interests A.R. is a scientific consultant for LifeMine Therapeutics, Inc. and a member of the Advisory Board of Current Biology. J.L.S. is a scientific consultant for Latch AI Inc., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The application of non-Saccharomyces yeasts in beer as a natural tool for innovation, to create different aroma profiles and flavoured non-alcoholic beers, has attracted great interest from both researchers and commercial brewers. As a result, a higher diversity of non-Saccharomyces yeasts for beer production is expected on the market in the coming years. However, the safe use of non-Saccharomyces yeasts has not been broadly investigated and no guidance for the safety assessment of yeasts is published. The fundamentals of a safety assessment include an accurate taxonomic species identification using up-to date methods, along with a literature study regarding the yeast species in question. The strain-specific safety concerns that should be assessed involve pathogenic potential, antifungal resistance, production of biogenic amines and possible allergic reactions. However, yeast safety assessment is in its infancy compared to bacterial safety assessment and research is needed to set cut-off values for antifungal resistance, identify potential virulence genes and validate screening tools to assess yeast strains. Finally, the individual breweries are responsible for the safety related to the process in which yeasts are applied and throughout the shelf life of the beer. The application of non-Saccharomyces yeasts for industrial beer production is promising in terms of defining new prototypes and developing healthier and safer beers, but only if good food safety measures, i.e., both for the strain and the production process, are in place throughout the food value chain. In this way, the ancient role of yeasts in making beverages safer and thereby improving food safety is emphasized., Competing Interests: Declaration of competing interest Most authors are employees at Chr. Hansen A/S, a major commercial supplier of starter cultures included yeast starter cultures for fermented beverage. Some of the authors are share-holders in Chr. Hansen A/S., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Formaldehyde and acetaldehyde are reactive small molecules produced endogenously in cells as well as being environmental contaminants. Both of these small aldehydes are classified as human carcinogens, since they are known to damage DNA and exposure is linked to cancer incidence. However, the mutagenic properties of formaldehyde and acetaldehyde remain incompletely understood, at least in part because they are relatively weak mutagens. Here, we use a highly sensitive yeast genetic reporter system featuring controlled generation of long single-stranded DNA regions to show that both small aldehydes induced mutational patterns characterized by predominantly C/G → A/T, C/G → T/A, and T/A → C/G substitutions, each in similar proportions. We observed an excess of C/G → A/T transversions when compared to mock-treated controls. Many of these C/G → A/T transversions occurred at TC/GA motifs. Interestingly, the formaldehyde mutational pattern resembles single base substitution signature 40 from the Catalog of Somatic Mutations in Cancer. Single base substitution signature 40 is a mutational signature of unknown etiology. We also noted that acetaldehyde treatment caused an excess of deletion events longer than 4 bases while formaldehyde did not. This latter result could be another distinguishing feature between the mutational patterns of these simple aldehydes. These findings shed new light on the characteristics of 2 important, commonly occurring mutagens., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Yeasts are increasingly employed in synthetic biology as chassis strains, including conventional and non-conventional species. It is still unclear how genomic evolution determines metabolic diversity among various yeast species and strains. In this study, we constructed draft GEMs for 332 yeast species using two alternative procedures from the toolbox RAVEN v 2.0. We found that draft GEMs could reflect the difference in yeast metabolic potentials, and therefore, could be utilized to probe the evolutionary trend of metabolism among 332 yeast species. We created a pan-draft metabolic model to account for the metabolic capacity of every sequenced yeast species by merging all draft GEMs. Further analysis showed that the pan-reactome of yeast has a "closed" property, which confirmed the great conservatism that exists in yeast metabolic evolution. Lastly, the quantitative correlations among trait similarity, evolutionary distances, genotype, and model similarity were thoroughly investigated. The results suggest that the evolutionary distance and genotype, to some extent, determine model similarity, but not trait similarity, indicating that multiple mechanisms shape yeast trait evolution. A large-scale reconstruction and integrative analysis of yeast draft GEMs would be a valuable resource to probe the evolutionary mechanism behind yeast trait variety and to further refine the existing yeast species-specific GEMs for the community.

The phyllosphere (i.e., the aerial parts of plants) harbors a rich microbial life, including bacteria, fungi, viruses, and yeasts. Current knowledge of yeasts stems primarily from industrial and medical research on Saccharomyces cerevisiae and Candida albicans, both of which can be found on plant tissues. For most other yeasts found in the phyllosphere, little is known about their ecology and functions. Here, we explore the diversity, dynamics, interactions, and genomics of yeasts associated with plant leaves and how tools and approaches developed for model yeasts can be adopted to disentangle the ecology and natural functions of phyllosphere yeasts. A first genomic survey exemplifies that we have only scratched the surface of the largely unexplored functional potential of phyllosphere yeasts., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Prospection of yeasts from oenological environments can provide knowledge of new native strains that are capable of fermenting must and positively influence the composition and sensory characteristics of the wine. This work addressed the biotechnological characterization of indigenous yeasts of Tannat must, an emblematic and widespread vineyard of Uruguay. Fifty-three yeast isolates were morphologically characterized and further identified by amplification and sequencing of ITS and D1-D2 regions, grouping into a total of fifteen species. One isolate of each species was randomly chosen and evaluated for its technological traits. In presence of ethanol (6 to 16% v/v) and sulfur dioxide (40 mg/L), native Saccharomyces cerevisiae 3FS presented the best growth rates and minor lag phase. Regarding non-Saccharomyces strains, Starmerella bacillaris 3MS stood out for its behavior in vinification conditions, more closely related to S. cerevisiae strains. Saccharomyces cerevisiae 3FS, Starmerella bacillaris 3MS, and Saturnispora diversa 1FS conducted a successful fermentation process reaching a final ethanol concentration ≥ 10% v/v and presenting a killer and resistant phenotype, suggesting that they could be used as pure starter cultures, as well as in mixed culture fermentations. This preliminary screening and oenological characterization of indigenous Saccharomyces cerevisiae and non-Saccharomyces yeasts might be a useful tool to identify some strains as potential candidates for wine vinification., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Oleaginous yeasts are typically defined as those able to accumulate more than 20% of their cell dry weight as lipids or triacylglycerides. Research on these yeasts has increased lately fuelled by an interest to use biotechnology to produce lipids and oleochemicals that can substitute those coming from fossil fuels or offer sustainable alternatives to traditional extractions (e.g., palm oil). Some oleaginous yeasts are attracting attention both in research and industry, with Yarrowia lipolytica one of the best-known and studied ones. Oleaginous yeasts can be found across several clades and different metabolic adaptations have been found, affecting not only fatty acid and neutral lipid synthesis, but also lipid particle stability and degradation. Recently, many novel oleaginous yeasts are being discovered, including oleaginous strains of the traditionally considered non-oleaginous Saccharomyces cerevisiae. In the face of this boom, a closer analysis of the definition of "oleaginous yeast" reveals that this term has instrumental value for biotechnology, while it does not give information about distinct types of yeasts. Having this perspective in mind, we propose to expand the term "oleaginous yeast" to those able to produce either intracellular or extracellular lipids, not limited to triacylglycerides, in at least one growth condition (including ex novo lipid synthesis). Finally, a critical look at Y. lipolytica as a model for oleaginous yeasts shows that the term "oleaginous" should be reserved only for strains and not species and that in the case of Y. lipolytica, it is necessary to distinguish clearly between the lipophilic and oleaginous phenotype., (© 2022 John Wiley & Sons Ltd.)

Microbes and their carbohydrate-active enzymes are central for depolymerization of complex lignocellulosic polysaccharides in the global carbon cycle. Their unique abilities to degrade and ferment carbohydrates are also utilized in many industrial processes such as baking, brewing and production of biofuels and drugs. Effective degradation and utilization of cellulose and hemicelluloses is important for the shift towards green bioeconomy, and requires microbes equipped with proper sets of carbohydrate-active enzymes (CAZymes). Knowledge of cellulolytic and xylanolytic CAZymes has mainly been generated from bacteria and filamentous fungi, while yeasts have been largely overlooked and may represent an untapped resource in natural CAZymes with industrial relevance. Cellulose and xylan-degrading yeasts with the ability to ferment saccharides are also promising candidates for consolidated bioprocesses (CBPs), as they can degrade lignocellulose and utilize its constituents to produce desired products at the same time. Cellulolytic yeasts able to utilize insoluble crystalline cellulose are rare while xylanolytic yeasts are rather widespread in nature. The lack of particular enzymes in yeasts can be remediated by introducing the missing enzymes into strains having outstanding product-forming attributes. In this review, we provide a comprehensive overview of the cellulose- and xylan-degrading ascomycetous and basidiomycetous yeasts known to date. We describe how these yeasts can be identified through bioprospecting and bioinformatic approaches and summarize available growth and enzymatic assays for strain characterization. Known and predicted CAZymes are extensively analyzed, both in individual species and in a phylogenetic perspective. We also describe the strategies used for construction of recombinant cellulolytic and xylanolytic strains as well as current applications for polysaccharide-degrading yeasts. Finally, we discuss the great potential of these yeasts as industrial cell factories, identify open research questions and provide suggestions for future investigations., (Copyright © 2022. Published by Elsevier Inc.)

Exopolysaccharides (EPS), metabolites of the medicinal edible fungus Monascus purpureus, have antioxidant, immunomodulatory, and anti-inflammatory effects. However, the biosynthetic mechanism of EPS from M. purpureus is still unclear, which hinders its utilization. In this study, the fermentation conditions of M. purpureus were optimized and comparative transcriptomic analysis was performed to understand the mechanisms and effects of fermentation on EPS synthesis. The optimal medium composition was 40 g/L mannose, 4 g/L yeast powder, 1 g/L MgSO 4 ·7H 2 O, 0.8 g/L KH 2 PO 4 , 1.6 g/L K 2 HPO 4 ·3H 2 O, and 2 mL/L Tween 80, and the optimal cultivation conditions were an inoculum of 7 %, culture temperature 30 °C, initial pH 6.0, and 180 rpm for 4 d. A total of 8095 unigenes were obtained, and 17 key enzymes for EPS synthesis were identified. Interestingly, 12 carbohydrate metabolism subcategories were enriched in the group with 4 days of fermentation compared to 2 days, with most of the differentially expressed genes (DEGs) being upregulated, but only nine carbohydrate metabolism subcategories were enriched with longer fermentation time, with all DEGs being downregulated. This study provides a theoretical basis for enhancing the EPS content and reveals the dynamics of EPS synthesis in M. purpureus, providing important targets for future EPS molecular modifications and gene knockdown studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Τhe aim of this study was to investigate the microbiological and physicochemical changes of pitted green olives (cvs. Conservolea and Halkidiki) elaborated in the Spanish style method and packaged in two types of multi-layered pouches under nitrogen atmosphere (100% N 2 ) for a period of 12 months. Moreover, the evolution of microbial consortia at the beginning (0 days), middle (180 days) and final (360 days) time points of storage was elucidated by plating, genotyped and identified through RAPD-PCR and ITS region amplicon sequencing, respectively, and subsequently metataxonomic analysis (for fungal communities only). Results showed that no enterobacteria could be detected on olive drupes, whereas the dominant microbiota from the onset of storage in both pouches consisted of LAB in populations ranging between ca. 4.2-6.6 log CFU/g. Although yeasts were initially enumerated at ca. 5.5 log CFU/g, they declined rapidly and could not be detected by plate counting after 30 days. The pH values increased from 4.11 to 4.24 and 4.03 to 4.12 at the beginning and end of storage for cvs. Halkidiki and Conservolea green olives, respectively. The total color difference index (ΔΕ*) presented a perceivable change in visual color (ΔΕ* > 2.0) only in cv. Conservolea olives during storage. Finally, molecular fingerprinting RAPD-PCR and 16S amplicon based identification revealed the dominance of five LAB species, namely Pediococcus ethanolidurans, Lactiplantibacillus pentosus, Lentilactobacillus rapi, Lentilactobacillus parafarraginis and Lentilactobacillus buchneri. Regarding the identification of yeasts, the metagenetic amplicon sequencing approach revealed fungal complexity in the olive samples. Pichia manshurica and Pichia membranifaciens prevailed during the first and middle stages of storage, whereas at the final stage higher complexity was noticeable., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mating disruption with insect sex pheromones is an attractive and environmentally friendly technique for pest management. Several Lepidoptera sex pheromones have been produced in yeast, where biosynthesis could be accomplished by the expression of fatty acyl-CoA desaturases and fatty acyl-CoA reductases. In this study, we aimed to develop yeast Yarrowia lipolytica cell factories for producing Lepidoptera pheromones which biosynthesis additionally requires β-oxidation, such as (Z)-7-dodecenol (Z7-12:OH), (Z)-9-dodecenol (Z9-12:OH), and (Z)-7-tetradecenol (Z7-14:OH). We expressed fatty acyl-CoA desaturases from Drosophila melanogaster (Dmd9) or Lobesia botrana (Lbo_PPTQ) and fatty acyl-CoA reductase from Helicoverpa armigera (HarFAR) in combinations with 11 peroxisomal oxidases of different origins. Yeast cultivations were performed with supplementation of methyl myristate (14:Me). The oxidase Lbo_31670 from L. botrana provided the highest titers of (Z)-7-dodecenoate, (Z)-9-dodecenoate, and (Z)-7-tetradecenoate. However, no chain-shortened fatty alcohols were produced. The mutation of fatty acid synthase (Fas2pI1220F) to increase myristate production did not lead to targeted fatty alcohol production. The problem was solved by directing the reductase into peroxisomes, where the strain with Dmd9 produced 0.10 ± 0.02 mg/l of Z7-12:OH and 0.48 ± 0.03 mg/l of Z7-14:OH, while the strain with Lbo_PPTQ produced 0.21 ± 0.03 mg/l of Z9-12:OH and 0.40 ± 0.07 mg/l of Z7-14:OH. In summary, the engineering of β-oxidation in Y. lipolytica allowed expanding the portfolio of microbially produced insect sex pheromones., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

White colony-forming yeasts (WCFYs) have been reported to form a white colony on the surface of kimchi, resulting in the deterioration of kimchi sensory quality. However, toxicity of WCFY has rarely been studied. Thus, to evaluate the safety of WCFY (i.e., Kazachstania servazzii, Candia sake, and Pichia kudriavzevii), we conducted cell and animal experiments as well as genomic analysis. In vitro studies indicated that WCFY did not induce cytotoxic responses such as lactate dehydrogenase release, excessive oxidative stress, and mitochondrial damage at concentrations of up to 2.5 × 10 5  CFU/mL in human intestinal and liver cells. In animal studies using rats (single-dose and 14-day repeated-dose oral toxicity studies), WCFY did not induce death, clinical signs of toxicity, histological alterations of the liver, or increases in the expression of pro-inflammatory cytokines nor cytochrome P450-2E1 in liver tissue at concentrations of up to 5 × 10 8  CFU/head/day. Genomic analysis revealed that P. kudriavzevii did not harbor genes related to toxicity and antimicrobial resistance. Taken together, our data suggest that exposure to WCFY through kimchi intake did not induce toxic response in the Caco-2, HepG2, and Sprague-Dawley rats. The current work provides evidence for the safety of accidental major WCFY ingestion via kimchi., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nonconventional yeasts have an untapped potential to expand biotechnology and enable process development necessary for a circular economy. They are especially convenient for the field of lipid and hydrocarbon biotechnology because they offer faster growth than plants and easier scalability than microalgae and exhibit increased tolerance relative to some bacteria. The ability of industrial organisms to import and metabolically transform lipids and hydrocarbons is crucial in such applications. Here, we assessed the ability of 14 yeasts to utilize 18 model lipids and hydrocarbons from six functional groups and three carbon chain lengths. The studied strains covered 12 genera from nine families. Nine nonconventional yeasts performed better than Saccharomyces cerevisiae, the most common industrial yeast. Rhodotorula toruloides, Candida maltosa, Scheffersomyces stipitis, and Yarrowia lipolytica were observed to grow significantly better and on more types of lipids and lipid molecules than other strains. They were all able to utilize mid- to long-chain fatty acids, fatty alcohols, alkanes, alkenes, and dicarboxylic acids, including 28 previously unreported substrates across the four yeasts. Interestingly, a phylogenetic analysis showed a short evolutionary distance between the R. toruloides, C. maltosa, and S. stipitis, even though R. toruloides is classified under a different phylum. This work provides valuable insight into the lipid substrate range of nonconventional yeasts that can inform species selection decisions and viability of lipid feedstocks., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Plants and microbes share common metabolic pathways for producing a range of bioproducts that are potentially foundational to the future bioeconomy. However, in planta accumulation and microbial production of bioproducts have never been systematically compared on an economic basis to identify optimal routes of production. A detailed technoeconomic analysis of four exemplar compounds (4-hydroxybenzoic acid [4-HBA], catechol, muconic acid, and 2-pyrone-4,6-dicarboxylic acid [PDC]) is conducted with the highest reported yields and accumulation rates to identify economically advantaged platforms and breakeven targets for plants and microbes. The results indicate that in planta mass accumulation ranging from 0.1 to 0.3 dry weight % (dwt%) can achieve costs comparable to microbial routes operating at 40 to 55% of maximum theoretical yields. These yields and accumulation rates are sufficient to be cost competitive if the products are sold at market prices consistent with specialty chemicals ($20 to $50/kg). Prices consistent with commodity chemicals will require an order-of-magnitude-greater accumulation rate for plants and/or yields nearing theoretical maxima for microbial production platforms. This comparative analysis revealed that the demonstrated accumulation rates of 4-HBA (3.2 dwt%) and PDC (3.0 dwt%) in engineered plants vastly outperform microbial routes, even if microbial platforms were to reach theoretical maximum yields. Their recovery and sale as part of a lignocellulosic biorefinery could enable biofuel prices to be competitive with petroleum. Muconic acid and catechol, in contrast, are currently more attractive when produced microbially using a sugar feedstock. Ultimately, both platforms can play an important role in replacing fossil-derived products.

In this study, we aimed to analyse the spoilage potential of the isolated yeast, LAB and AAB species. Thus, 11 strains were inoculated at 0·3% (v/v) into a sterile filtered tchapalo and stored for 3 days at ambient temperature (27-30°C). All the tested strains grew well or remained stable except for Limosilactobacillus fermentum and Pediococcus acidilactici, which decreased throughout the storage time. A significant decrease of total soluble solids was observed only for Saccharomyces cerevisiae (from 7·8 to 5·8 °Brix) and Meyerozyma guilliermondii (from 7·8 to 5·5 °Brix). The tchapalo samples inoculated with the LAB strains Weissella paramesenteroides, P. acidilactici, L. fermentum and the yeast strain Candida tropicalis were judged similar to the control by the panellists. However, the strains of Lacticaseibacillus paracasei and Latilactobacillus curvatus (LAB), S. cerevisiae, M. guilliermondii and Kluyveromyces marxianus (yeasts) and Acetobacter pasteurianus and A. cerevisiae (AAB) induced the spoilage of the tchapalo appearance, smell and/or taste. In the spoiled tchapalo quantitative and qualitative modification of some volatile compounds (VOCs), such as lilac aldehyde, ethyl acetate, ethyl hexanoate, ethyl octanoate and phenethyl acetate, were observed. These results provide information about the microorganisms that need to be removed to extend the shelf life of tchapalo., (© 2022 The Society for Applied Microbiology.)

Reptiles have become popular exotic pets and in some parts of the world, they are used as important source of food, medicines, and materials. Synanthropic lizards are recognized as reservoirs of viruses, bacteria, and parasites but their role in dissemination of zoonotic pathogenic yeasts in the environment was never investigated. Therefore, fecal samples (n=177) from Podarcis siculus (Italian wall lizard), Chalcides ocellatus (Ocellated skink) and Tarentola mauritanica (Moorish gecko) were collected and yeasts were isolated and identified biochemically and molecularly by sequencing the rDNA internal transcribed spacer region (ITS). The phylogenetical relationship of isolated yeast species and their antifungal susceptibility profiles to ten antifungal agents were also assessed. Sixty samples (n=60/177; 33.9%) scored positive for yeasts, with the highest occurrence in C. ocellatus (n=11/17; 64.7%) and the highest variety of species in P. siculus (n=11/12; 91.6%). A total of 364 isolates belonging to Candida, Trichosporon, Saccharomyces and Geotrichum genera were molecularly identified. In particular, Candida albicans (n=160; 44%) followed by Trichosporon coremiiforme (n=44; 12.1%), Pichia kudriavzevii (n=32; 8.8%) and Trichosporon asahii (n=28; 7.7%) were the most frequently isolated species. The phylogenetic tree grouped all representative sequence types within the clade including Candida spp. strains from different geographical areas and from animal species, including human. All tested strains showed high susceptibility to the assayed antifungal drugs. This study suggests the role of lizards as reservoirs and spreaders of zoonotic pathogenic yeasts in the environment. The absence of resistance phenomena in the isolated yeasts might reflect an environment free of azole antifungal pollution or chemicals, suggesting the usefulness of these animals as bio indicators of environment quality., (Copyright © 2022. Published by Elsevier B.V.)

Peptidoglycan (PG) was targeted as the marker for bacterial occurrence inside yeast. Detection of only few bacteria in old and new generations of yeast raised the question of how yeast controls the abundance of its intracellular bacteria. One gastric C. tropicalis that showed concurrence of H. pylori and Staphylococcus 16S rDNA was stained for assessing the viability of intracellular bacteria. Fluorescein isothiocyanate (FITC)-labeled anti-PG monoclonal antibody (APGMAb) was used for detection of PG inside yeast by direct immunofluorescence. APGMAb-coated magnetic beads were used for separation of bacteria from disrupted yeasts. Bead-bound bacteria were separated, fixed, stained, and examined by scanning electron microscope (SEM). Bead-bound bacteria were cultured and identified by amplification and sequencing of 16S rDNA. Fluorescence microscopy demonstrated occurrence of few live bacteria inside yeast cells. FITC- APGMAb interacted with PG of intracellular bacteria, appearing as few green spots in mother and daughter yeast cells. Interestingly, PG fragments were also detected in the exterior of yeast cells. SEM observations showed separated bead-bound bacilli and cocci. Culture of Staphylococcus was positive. Sequencing results confirmed identity of separated bacteria as H. pylori and Staphylococcus. PG detected inside yeast may have belonged to H. pylori, Staphylococcus or any other intracellular bacteria that coexisted in yeast as its microbiome. Detection of only few intracellular bacteria in old and new generations of yeast as well as PG fragments in their exterior suggested that yeast controls the abundance of its intracellular bacteria at low rate by hydrolysis and exporting of PG., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

For decades, budding yeast, a single-cellular eukaryote, has provided remarkable insights into human biology. Yeast and humans share several thousand genes despite morphological and cellular differences and over a billion years of separate evolution. These genes encode critical cellular processes, the failure of which in humans results in disease. Although recent developments in genome engineering of mammalian cells permit genetic assays in human cell lines, there is still a need to develop biological reagents to study human disease variants in a high-throughput manner. Many protein-coding human genes can successfully substitute for their yeast equivalents and sustain yeast growth, thus opening up doors for developing direct assays of human gene function in a tractable system referred to as 'humanized yeast'. Humanized yeast permits the discovery of new human biology by measuring human protein activity in a simplified organismal context. This Review summarizes recent developments showing how humanized yeast can directly assay human gene function and explore variant effects at scale. Thus, by extending the 'awesome power of yeast genetics' to study human biology, humanizing yeast reinforces the high relevance of evolutionarily distant model organisms to explore human gene evolution, function and disease., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Wildlife animals are recognized as reservoirs for zoonotic fungi and their faeces might play an important role in introducing pathogens into the environment. Thought wild boar (Sus scrofa) population has dramatically increased across Europe, information about their possible role in dissemination of zoonotic pathogenic yeasts in the environment is scant. Therefore, fecal samples (n = 124) from wild boars from Campania region (Southern Italy) were collected and yeasts identified biochemically and molecularly by sequencing of the internal transcribed spacer region and their phylogenetical relationship assessed. The antifungal susceptibility profiles of yeasts were also investigated using AFST-EUCAST method. Yeasts were isolated from 50.1% of the samples with the highest occurrence in samples from the province of Salerno (61.1%). A total of 368 Candida strains belonging to nine species were identified, with Candida albicans (45.7%), followed by Candida krusei (15.2%), Kazachstania slooffiae (9.8%) and Candida parapsilosis (7.6%) as the most prevalent identified species. Among C. albicans four sequence types (i.e., ST1-ST4) were identified with an intraspecific nucleotide difference up to 0.21%. The ML tree grouped all representative sequence types as paraphyletic clades with those of the references yeast species, respectively and supported by high bootstrap values. Fluconazole was the less active drug whereas, posaconazole, voriconazole, and isavuconazole the most active one. No resistance phenomena were observed for C. albicans and high MICs values for 5FC, azoles and echinocandines were registered in non-albicans Candida spp. This study showed, for the first time, the important role of wild boars in dissemination of pathogenic fungi in the environment. The absence of resistance phenomena in the Candida spp. might reflect environmental free from residues of azoles antifungals pollution or chemicals and suggests the role of wild boar as bio indicators of environment quality., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Methylotrophic yeasts have been widely recognized as a promising host for production of recombinant proteins and value-added chemicals. Promoters for controlled gene expression are critical for construction of efficient methylotrophic yeasts cell factories. Here, we summarized recent advances in characterizing and engineering promoters in methylotrophic yeasts, such as Komagataella phaffii and Ogataea polymorpha. Constitutive and inducible promoters controlled by methanol or other inducers/repressors were introduced to demonstrate their applications in production of proteins and chemicals. Furthermore, efforts of promoter engineering, including site-directed mutagenesis, hybrid promoter, and transcription factor regulation to expand the promoter toolbox were also summarized. This mini-review also provides useful information on promoters for the application of metabolic engineering in methylotrophic yeasts. KEY POINTS: • The characteristics of six methylotrophic yeasts and their promoters are described. • The applications of Komagataella phaffii and Ogataea polymorpha in metabolic engineeringare expounded. • Three promoter engineering strategies are introduced in order to expand the promoter toolbox., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims: The yeast community structure associated with grapes is an essential part of the wine-growing chain with a significant effect on wine quality. The aim of the present study was to evaluate the effect of the varietal factor on the yeast community assembly on grapes and during must fermentation., Methods and Results: We analysed the wine yeast populations associated with four different grape varieties from the Greek national collection vineyard of Lykovryssi. The vintage effect was also considered by sampling the grapes for two consecutive years. Fourteen yeast species were recovered and genotyped to distinct subpopulations. A relatively stable yeast community structure was detected across vintages, with Hanseniaspora guilliermondii being the core species of the vineyard under study. The detected species subpopulations shared a relatively high genetic similarity with several genotypes persisting across vintages., Conclusions: It was shown that different grape cultivars were associated with distinct yeast communities, pointing to their possible implication on wine chemical diversity., Significance and Impact of the Study: Present findings show that the varietal factor is an important sharpener of the vineyard-associated wine yeast community, which may interfere with the organoleptic profile of the resulting wines., (© 2022 Society for Applied Microbiology.)

Yeasts isolated from the worker caste of the Colombian leaf-cutting ant, Atta cephalotes (Hymenoptera: Myrmicinae) were cultured and identified by molecular methods. Abundant, persistent, and omnipresent species were classified as "prevalent". Experimental data were compared with information gathered from published reports on the yeast species composition in other leaf-cutting ant species. Diversity analysis was conducted using diversity values (q0, q1, and q2) to compare the richness and abundance of yeasts present in different leaf-cutting ant species. Clustering analysis was carried out to assess the similarity of yeast community according to ant species. The yeast species composition was highly variable among the ant species. A. laevigata and A. capiguara showed the highest degree of similarity and differed from the group composed by A. cephalotes, A. sexdens, A. sexdens rubropilosa, and A. texana. The isolation of dominant yeasts in different ant castes within the different compartments of a colony strongly suggests that the identified microorganisms are not transient but are native to the soil surrounding ant colonies and the substrates used by the ants to grow their fungal cultivars. It is apparent that the ant-fungus mutualism does not operate in an environment devoid of other microbes, but rather that the association must be seen within the context of a background of other microorganisms, particularly the dominant yeasts., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

As essential structural molecules of fungal cell membrane, ergosterol is not only an important component of fungal growth and stress-resistance but also a key precursor for manufacturing steroid drugs of pharmaceutical or agricultural significance. So far, ergosterol biosynthesis in yeast has been elucidated elaborately, and efforts have been made to increase ergosterol production through regulation of ergosterol metabolism and storage. Furthermore, the same intermediates shared by yeasts and animals or plants make the construction of heterologous sterol pathways in yeast a promising approach to synthesize valuable steroids, such as phytosteroids and animal steroid hormones. During these challenging processes, several obstacles have arisen and been combated with great endeavors. This paper reviews recent research progress of yeast metabolic engineering for improving the production of ergosterol and heterologous steroids. The remaining tactics are also discussed., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Gene editing technology plays an extremely significant role in synthetic biology and metabolic engineering. Traditional genetic manipulation methods, such as homologous recombination, however, are inefficient, time-consuming, and barely feasible when disrupting multiple genes simultaneously. Starmerella bombicola , a nonconventional yeast that overproduces sophorolipids, lacks convenient genetic tools for engineering strains. Here, we developed an efficient CRISPR-Cas9 genome editing technology by combining molecular element mining and expression system optimization for S. bombicola . This CRISPR-Cas9 system improved the efficiency of gene-integration/target gene-introducing disruption by homology-directed repair and realized the multi-gene simultaneous disruptions. Based on this CRISPR-Cas9 system, we also further constructed an engineered strain via the in vivo assembly of multiple DNA fragments (10 kb) that can produce acid-type sophorolipids. These results showed that the CRISPR-Cas9 system may be an efficient and convenient strategy to perform genetic manipulation in S. bombicola .

For the past few decades, industrialization has made a huge environmental hazard to the world with its waste. The approach of waste to wealth in the recent era has made many Eco-economical suggestions for the industries. The valuable products in biorefinery aspects of the eco-economical suggestions include; energy products, high-value drugs and novel materials. Bio-lipids are found to be the major influencing eco-economical products in the process. Production of bio-lipid from microbial sources has paved the way for future research on lipid-bioproducts. The yeast cell is a unique organism with a large unicellular structure capable of accumulating a high amount of lipids. It constitutes 90% of neutral lipids. Various strategies enhance the lipid profile of yeast cells: usage of oleaginous yeast, usage of low cost (or) alternative substrates, developing stress conditions in the growth medium, using genetically modified yeast, altering metabolic pathways of yeast and by using the symbiotic cultures of yeast with other microbes. The metabolic alterations of lipid pathways such as lipid biosynthesis, lipid elongation, lipid accumulation and lipid degradation have been a striking feature of research in lipid-based microbial work. The lipid-bioproducts have also made a strong footprint in the history of alternative energy products. It includes partial acyl glycerol, oleochemicals, phospholipids and biofuels. This report comprises the recent approaches carried out in the yeast cell for enhancing its lipid content. The limitations, challenges and future scope of individual strategies were also highlighted in this article., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Yeasts play an important role in spontaneous fermentation of traditional alcoholic beverages. Our previous study revealed that a mixed-consortia of both Saccharomyces and non- Saccharomyces yeasts were responsible for fermentation of khadi , a popular, non-standardized traditional beverage with an immense potential for commercialization in Botswana. Functional characterization of isolated fermenting yeasts from mixed consortia is an indispensable step towards the selection of potential starter cultures for commercialization of khadi . In this study, we report the characterization of 13 khadi isolates for the presence of brewing-relevant phenotypes such as their fermentative capacity, ability to utilize a range of carbon sources and their ability to withstand brewing-associated stresses, as a principal step towards selection of starter cultures. Khadi isolates such as Saccharomyces cerevisiae , Saccharomycodes ludwigii and Candida ethanolica showed good brewing credentials but Lachancea fermentati emerged as the isolate with the best brewing attributes with a potential as a starter culture. However, we were then prompted to investigate the potential of L. fermentati to influence the fruity aromatic flavor, characteristic of khadi . The aroma components of 18 khadi samples were extracted using headspace solid phase micro-extraction (HSSPME) and identified using a GC-MS. We detected esters as the majority of volatile compounds in khadi , typical of the aromatic signature of both khadi and L. fermentati associated fermentations. This work shows that L. fermentati has potential for commercial production of khadi .

Microbial cell surface layers, which mainly include the cell membrane, cell wall, periplasmic space, outer membrane, capsules, S-layers, pili, and flagella, control material exchange between the cell and the extracellular environment, and have great impact on production titers and yields of various bio-products synthesized by microbes. Recent research work has made exciting achievements in metabolic engineering using microbial cell surface components as novel regulation targets without direct modifications of the metabolic pathways of the desired products. This review article will summarize the accomplishments obtained in this emerging field, and will describe various engineering strategies that have been adopted in bacteria and yeasts for the enhancement of mass transfer across the cell surface, improvement of protein expression and folding, modulation of cell size and shape, and re-direction of cellular resources, all of which contribute to the construction of more efficient microbial cell factories toward the synthesis of a variety of bio-products. The existing problems and possible future directions will also be discussed., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Yeasts play important roles in both the environment and in human welfare. While some environmental yeasts positively contribute to nutrient cycling and food production, a significant number of yeast species are opportunistic human pathogens, including several that are tolerant/resistant to commonly used antifungal drugs. At present, most of our understanding of environmental yeasts has come from a few terrestrial environments in selected geographic regions. Relatively little is known about yeast diversity in tropical environments and their potential impacts on human health. Here, we characterize culturable yeasts in 968 environmental samples from eight regions in tropical China. Among the 516 soil, 273 freshwater, and 179 seawater samples, 71.5%, 85.7%, and 43.6% contained yeasts, respectively. A total of 984 yeast isolates were analyzed for their DNA barcode sequences and their susceptibilities to fluconazole. DNA sequence comparisons revealed that the 984 yeast isolates likely belonged to 144 species, including 106 known species and 38 putative novel species. About 38% of the 984 isolates belonged to known human pathogens and the most common species was Candida tropicalis , accounting for 21% (207/984) of all isolates. Further analyses based on multi-locus sequence typing revealed that some of these environmental C. tropicalis shared identical genotypes with clinical isolates previously reported from tropical China and elsewhere. Importantly, 374 of the 984 (38%) yeast isolates showed intermediate susceptibility or resistance to fluconazole. Our results suggest that these environmental yeasts could have significant negative impacts on human health.

Soft drinks are food matrices propitious to the growth of acidophilic bacteria, yeasts, and filamentous fungi due to their pH, water activity, and the presence of nutrients. Off-flavor, clouding, and package stuffing are the only parameters producers have to detect spoilage when it is often too late for the brand's reputation. In this work, microbiological analyses were performed on non-alcoholic beverages of Brazilian and Bolivian brands. As a result, Gram-positive, Gram-negative, yeast, and filamentous fungi were isolated. Zygosaccharomyces bisporus yeast was isolated from different flavored stuffed products, and Gluconacetobacter liquefaciens and Brevibacillus agri were isolated from packages without visible signs of deterioration. These microorganisms were identified by MALDI-TOF. For products with visible growth of filamentous fungi, microscopic identification keys identified Aspergillus flavus, Penicillium citrinum, Paecilomyces niveus, and Paecilomyces variotii. These work's findings reflect a failure to sanitize raw materials since the isolates' primary origin is the soil and the water, pointing to the lack of process control in soft drinks., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The prevalence of multicellular organisms is due in part to their ability to form complex structures. How cells pack in these structures is a fundamental biophysical issue, underlying their functional properties. However, much remains unknown about how cell packing geometries arise, and how they are affected by random noise during growth - especially absent developmental programs. Here, we quantify the statistics of cellular neighborhoods of two different multicellular eukaryotes: lab-evolved 'snowflake' yeast and the green alga Volvox carteri . We find that despite large differences in cellular organization, the free space associated with individual cells in both organisms closely fits a modified gamma distribution, consistent with maximum entropy predictions originally developed for granular materials. This 'entropic' cellular packing ensures a degree of predictability despite noise, facilitating parent-offspring fidelity even in the absence of developmental regulation. Together with simulations of diverse growth morphologies, these results suggest that gamma-distributed cell neighborhood sizes are a general feature of multicellularity, arising from conserved statistics of cellular packing., Competing Interests: TD, SH, SZ, DY, AB, JP, AH, HW, HS, WR, PY No competing interests declared, RG Reviewing editor, eLife, (© 2022, Day et al.)

Genomic rearrangements often generate phenotypic diversification. We previously reported the TAQing system where genomic rearrangements are induced via conditional activation of a restriction endonuclease in yeast and plant cells to produce mutants with marked phenotypic changes. Here we developed the TAQing2.0 system based on the direct delivery of endonucleases into the cell nucleus by cell-penetrating peptides. Using the optimized procedure, we introduce a heat-reactivatable endonuclease TaqI into an asexual industrial yeast (torula yeast), followed by a transient heat activation of TaqI. TAQing2.0 leads to generation of mutants with altered flocculation and morphological phenotypes, which exhibit changes in chromosomal size. Genome resequencing suggested that torula yeast is triploid with six chromosomes and the mutants have multiple rearrangements including translocations having the TaqI recognition sequence at the break points. Thus, TAQing2.0 is expected as a useful method to obtain various mutants with altered phenotypes without introducing foreign DNA into asexual industrial microorganisms., (© 2022. The Author(s).)

When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of the relevant cellular constraints remain to be determined for such cells. Here, we present a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions. The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data suggests that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation-known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimisation, providing a framework to understand metabolic adaptation in eukaryal cells., (© 2022. The Author(s).)

High-throughput chromosome conformation capture (Hi-C) has recently been applied to natural microbial communities and revealed great potential to study multiple genomes simultaneously. Several extraneous factors may influence chromosomal contacts rendering the normalization of Hi-C contact maps essential for downstream analyses. However, the current paucity of metagenomic Hi-C normalization methods and the ignorance for spurious interspecies contacts weaken the interpretability of the data. Here, we report on two types of biases in metagenomic Hi-C experiments: explicit biases and implicit biases, and introduce HiCzin, a parametric model to correct both types of biases and remove spurious interspecies contacts. We demonstrate that the normalized metagenomic Hi-C contact maps by HiCzin result in lower biases, higher capability to detect spurious contacts, and better performance in metagenomic contig clustering.

Mitochondria are essential to cell homeostasis, and alterations in mitochondrial distribution, segregation, and turnover have been linked to complex pathologies such as neurodegenerative diseases and cancer. Understanding how these functions are coordinated in specific cell types is a major challenge to discover how mitochondria globally shape cell functionality. In this review, we first describe how mitochondrial transport and dynamics are regulated throughout the cell cycle in yeast and in mammals. Second, we explore the functional consequences of mitochondrial transport and partitioning on cell proliferation, fate acquisition, and stemness and on the way cells adapt their metabolism. Finally, we focus on how mitochondrial clearance programs represent a further layer of complexity for cell differentiation or in the maintenance of stemness. Defining how mitochondrial transport, dynamics, and clearance are mutually orchestrated in specific cell types may help our understanding of how cells can transition from a physiological to a pathological state.

Aims: Isolation, characterization and assessment of inhibitor tolerance of thermotolerant yeasts associated with distillery effluent and molasses, and their use in high-temperature ethanol production from alkali-treated rice straw., Methods and Results: A total of 92 thermotolerant yeasts were isolated from seven different distillery effluent and molasses samples. Based on MSP-PCR, 34 yeasts were selected and identified by sequencing the D1/D2 domain of LSU rDNA. These yeasts belonged to eight genera and nine different species. We assessed the inhibitor tolerance of these 34 well-characterized yeasts against various pre-treatment-generated inhibitors (furfural, 5-hydroxymethyl furfural and acetic acid) and also evaluated their ethanol yields at 40, 45 and 50℃. Among selected strains, Pichia kudriavzevii DSA3.2 exhibited the highest ethanol production (24.5 g l -1 ) with an efficiency of 95.7% at 40℃ using 5% glucose. At 45℃, P. kudriavzevii DSA3.2 and Kluyveromyces marxianus MSS6.3 yielded maximum ethanol titres; 22.3 and 23 g l -1 with 87.4% and 90% efficiency, respectively. While using alkali-treated RS at 45℃, K. marxianus MSS6.3 produced 10.5 g l -1 of ethanol with 84.5% fermentation efficiency via separate hydrolysis and fermentation, and 10.9 g l -1 of ethanol with 85% efficiency via simultaneous saccharification and fermentation. Pichia kudriavzevii DSA3.2, DSA3.1 and K. marxianus MSS6.3 also exhibited significant tolerance against multiple inhibitors., Conclusions: Yeast isolates P. kudriavzevii DSA3.2 and K. marxianus MSS6.3 exhibited significant inhibitor tolerance and proved to be suitable for high-temperature ethanol fermentation. After additional optimization and scale-up experiments, these isolates can be exemplary candidates for industrial-scale ethanol production from lignocellulosic biomass., Significance and Impact of the Study: Our study recognizes distillery effluents and molasses as specialized niches for yeasts with a broad substrate range, capable of tolerating multiple inhibitors and yielding high levels of ethanol at elevated temperatures. These yeasts can further be exploited for bioethanol production through SSF/SHF at a larger scale., (© 2021 Society for Applied Microbiology.)

Cost-effective microbial conversion processes of renewable feedstock into biofuels and biochemicals are of utmost importance for the establishment of a robust bioeconomy. Conventional baker's yeast Saccharomyces cerevisiae, widely employed in biotechnology for decades, lacks many of the desired traits for such bioprocesses like utilization of complex carbon sources or low tolerance towards challenging conditions. Many non-conventional yeasts (NCY) present these capabilities, and they are therefore forecasted to play key roles in future biotechnological production processes. For successful implementation of NCY in biotechnology, several challenges including generation of alternative carbon sources, development of tailored NCY and optimization of the fermentation conditions are crucial for maximizing bioproduct yields and titers. Addressing these challenges requires a multidisciplinary approach that is facilitated through the 'YEAST4BIO' COST action. YEAST4BIO fosters integrative investigations aimed at filling knowledge gaps and excelling research and innovation, which can improve biotechnological conversion processes from renewable resources to mitigate climate change and boost transition towards a circular bioeconomy. In this perspective, the main challenges and research efforts within YEAST4BIO are discussed, highlighting the importance of collaboration and knowledge exchange for progression in this research field., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Cells have compensatory mechanisms to coordinate the rates of major biological processes, thereby permitting growth in a wide variety of conditions. Here, we uncover a compensatory link between cleavage/polyadenylation in the nucleus and messenger RNA (mRNA) turnover in the cytoplasm. On a global basis, same-gene 3' mRNA isoforms with twofold or greater differences in half-lives have steady-state mRNA levels that differ by significantly less than a factor of 2. In addition, increased efficiency of cleavage/polyadenylation at a specific site is associated with reduced stability of the corresponding 3' mRNA isoform. This inverse relationship between cleavage/polyadenylation and mRNA isoform half-life reduces the variability in the steady-state levels of mRNA isoforms, and it occurs in all four growth conditions tested. These observations suggest that during cleavage/polyadenylation in the nucleus, mRNA isoforms are marked in a manner that persists upon translocation to the cytoplasm and affects the activity of mRNA degradation machinery, thus influencing mRNA stability., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

4α-Glucanotransferase (4α-GTase) is unique in its ability to form cyclic oligosaccharides, some of which are of industrial importance. Generally, low amount of enzymes is produced by or isolated from their natural sources: animals, plants, and microorganisms. Heterologous expressions of these enzymes, in an attempt to increase their production for applicable uses, have been widely studied since 1980s; however, the expressions are mostly performed in the prokaryotic bacteria, mostly Escherichia coli. Site-directed mutagenesis has added more value to these expressed enzymes to display the desired properties beneficial for their applications. The search for further suitable properties for food application leads to an extended research in expression by another group of host organism, the generally-recognized as safe host including the Bacillus and the eukaryotic yeast systems. Herein, our review focuses on two types of 4α-GTase: the cyclodextrin glycosyltransferase and amylomaltase. The updated studies on the general structure and properties of the two enzymes with emphasis on heterologous expression, mutagenesis for property improvement, and their industrial applications are provided., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Natural hypersaline environments are inhabited by an abundance of prokaryotic and eukaryotic microorganisms capable of thriving under extreme saline conditions. Yeasts represent a substantial fraction of halotolerant eukaryotic microbiomes and are frequently isolated as food contaminants and from solar salterns. During the last years, a handful of new species has been discovered in moderate saline environments, including estuarine and deep-sea waters. Although Saccharomyces cerevisiae is considered the primary osmoadaptation model system for studies of hyperosmotic stress conditions, our increasing understanding of the physiology and molecular biology of halotolerant yeasts provides new insights into their distinct metabolic traits and provides novel and innovative opportunities for genome mining of biotechnologically relevant genes. Yeast species such as Debaryomyces hansenii, Zygosaccharomyces rouxii, Hortaea werneckii and Wallemia ichthyophaga show unique properties, which make them attractive for biotechnological applications. Select halotolerant yeasts are used in food processing and contribute to aromas and taste, while certain gene clusters are used in second generation biofuel production. Finally, both pharmaceutical and chemical industries benefit from applications of halotolerant yeasts as biocatalysts. This comprehensive review summarizes the most recent findings related to the biology of industrially-important halotolerant yeasts and provides a detailed and up-to-date description of modern halotolerant yeast-based biotechnological applications., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Optogenetics involves the use of light to control cellular functions and has become increasingly popular in various areas of research, especially in the precise control of gene expression. While this technology is already well established in neurobiology and basic research, its use in bioprocess development is still emerging. Some optogenetic switches have been implemented in yeasts for different purposes, taking advantage of a wide repertoire of biological parts and relatively easy genetic manipulation. In this review, we cover the current strategies used for the construction of yeast strains to be used in optogenetically controlled protein or metabolite production, as well as the operational aspects to be considered for the scale-up of this type of process. Finally, we discuss the main applications of optogenetic switches in yeast systems and highlight the main advantages and challenges of bioprocess development considering future directions for this field., (Copyright © 2021. Published by Elsevier Inc.)

Yeasts are ubiquitous in temperate forests. While this broad habitat is well-defined, the yeasts inhabiting it and their life cycles, niches, and contributions to ecosystem functioning are less understood. Yeasts are present on nearly all sampled substrates in temperate forests worldwide. They associate with soils, macroorganisms, and other habitats and no doubt contribute to broader ecosystem-wide processes. Researchers have gathered information leading to hypotheses about yeasts' niches and their life cycles based on physiological observations in the laboratory as well as genomic analyses, but the challenge remains to test these hypotheses in the forests themselves. Here, we summarize the habitat and global patterns of yeast diversity, give some information on a handful of well-studied temperate forest yeast genera, discuss the various strategies to isolate forest yeasts, and explain temperate forest yeasts' contributions to biotechnology. We close with a summary of the many future directions and outstanding questions facing researchers in temperate forest yeast ecology. Yeasts present an exciting opportunity to better understand the hidden world of microbial ecology in this threatened and global habitat., (© 2022 John Wiley & Sons, Ltd.)

Chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) is a powerful method to identify protein interactions, and has long been used to gain insights into regulatory networks in relevant fungal species as well as many other organisms. In this chapter, we discuss a similar technique called ChIP-SICAP (chromatin immunoprecipitation with selective isolation of chromatin-associated proteins) that overcomes many of the traditional limitations of ChIP-MS, and describe a protocol that allows ChIP-SICAP to be applied to Candida albicans and other yeasts. Notably, the technique design permits stringent washing to remove contaminating proteins and antibodies before subsequent mass spectrometry processing, allows for genome-wide mapping of the bait protein by ChIP-seq after ChIP-SICAP from the same sample through a DNA recovery process, and specifically purifies and identifies proteins associating with chromatin. In the future, ChIP-SICAP will provide the yeast genomics research community an additional method to explore the complex dynamics of the gene-regulatory networks modulating morphology, metabolism and response to stress., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Evolutionary engineering of microbes provides a powerful tool for untargeted optimization of (engineered) cell factories and identification of genetic targets for further research. Directed evolution is an intrinsically time-intensive effort, and automated methods can significantly reduce manual labor. Here, design considerations for various evolutionary engineering methods are described, and generic workflows for batch-, chemostat-, and accelerostat-based evolution in automated bioreactors are provided. These methods can be used to evolve yeast cultures for >1000 generations and are designed to require minimal manual intervention., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

In the beer brewing industry, microbial spoilage presents a consistent threat that must be monitored and controlled to ensure the palatability of a finished product. Many of the predominant beer spoilage microbes have been identified and characterized, but the mechanisms of contamination and persistence remain an open area of study. Postproduction, many beers are distributed as kegs that are attached to draft delivery systems in retail settings where ample opportunities for microbial spoilage are present. As such, restaurants and bars can experience substantial costs and downtime for cleaning when beer draft lines become heavily contaminated. Spoilage monitoring on the retail side of the beer industry is often overlooked, yet this arena may represent one of the largest threats to the profitability of a beer if its flavor profile becomes substantially distorted by contaminating microbes. In this study, we sampled and cultured microbial communities found in beers dispensed from a retail draft system to identify the contaminating bacteria and yeasts. We also evaluated their capability to establish new biofilms in a controlled setting. Among four tested beer types, we identified over a hundred different contaminant bacteria and nearly 20 wild yeasts. The culturing experiments demonstrated that most of these microbes were viable and capable of joining new biofilm communities. These data provide an important reference for monitoring specific beer spoilage microbes in draft systems and we provide suggestions for cleaning protocol improvements. IMPORTANCE Beer production, packaging, and service are each vulnerable to contamination by microbes that metabolize beer chemicals and impart undesirable flavors, which can result in the disposal of entire batches. Therefore, great effort is taken by brewmasters to reduce and monitor contamination during production and packaging. A commonly overlooked quality control stage of a beer supply chain is at the retail service end, where beer kegs supply draft lines in bars and restaurants under nonsterile conditions. We found that retail draft line contamination is rampant and that routine line cleaning methods are insufficient to efficiently suppress beer spoilage. Thus, many customers unknowingly consume spoiled versions of the beers they consume. This study identified the bacteria and yeast that were resident in retail draft beer samples and also investigated their abilities to colonize tubing material as members of biofilm communities.

Aerobic fermentation, also referred to as the Crabtree effect in yeast, is a well-studied phenomenon that allows many eukaryal cells to attain higher growth rates at high glucose availability. Not all yeasts exhibit the Crabtree effect, and it is not known why Crabtree-negative yeasts can grow at rates comparable to Crabtree-positive yeasts. Here, we quantitatively compared two Crabtree-positive yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe , and two Crabtree-negative yeasts, Kluyveromyces marxianus and Scheffersomyces stipitis , cultivated under glucose excess conditions. Combining physiological and proteome quantification with genome-scale metabolic modeling, we found that the two groups differ in energy metabolism and translation efficiency. In Crabtree-positive yeasts, the central carbon metabolism flux and proteome allocation favor a glucose utilization strategy minimizing proteome cost as proteins translation parameters, including ribosomal content and/or efficiency, are lower. Crabtree-negative yeasts, however, use a strategy of maximizing ATP yield, accompanied by higher protein translation parameters. Our analyses provide insight into the underlying reasons for the Crabtree effect, demonstrating a coupling to adaptations in both metabolism and protein translation., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

DNA lesions have properties that allow them to escape their nuclear compartment to achieve DNA repair in another one. Recent studies uncovered that the replication fork, when its progression is impaired, exhibits increased mobility when changing nuclear positioning and anchors to nuclear pore complexes, where specific types of homologous recombination pathways take place. In yeast models, increasing evidence points out that nuclear positioning is regulated by small ubiquitin-like modifier (SUMO) metabolism, which is pivotal to maintaining genome integrity at sites of replication stress. Here, we review how SUMO-based pathways are instrumental to spatially segregate the subsequent steps of homologous recombination during replication fork restart. In particular, we discussed how routing towards nuclear pore complex anchorage allows distinct homologous recombination pathways to take place at halted replication forks.

Floral nectar is colonised by microbes, especially yeasts which alter the scent, temperature, and chemical composition of nectar, thereby playing an essential role in pollination. The yeast communities inhabiting the nectar of tropical flowers of India are not well explored. We isolated 48 yeast strains from seven different tropical flowering plants. Post MSP-PCR-based screening, 23 yeast isolates and two yeast-like fungi were identified, which belonged to 16 species of 12 genera viz. Candida (2 species), Aureobasidium (2 species), Metschnikowia (2 species), Meyerozyma (1 species), Saitozyma (1 species), Wickerhamomyces (1 species), Kodamaea (2 species), Pseudozyma (1 species), Starmerella (1 species), Hanseniaspora (1 species), Rhodosporidiobolus (1 species), Moesziomyces (1 species), and two putative novel species. All yeast strains were assessed for their osmotolerance abilities in high salt and sugar concentration. Among all the isolates, C. nivariensis (SRA2.2, SRA1.1 and SRA2.1), M. caribbica (SRA4.8 and SRA4.6), S. flava SRA4.2, and M. reukaufii SRA3.2 showed significant growth in high concentrations of sugar (40-50% glucose), as well as salt (12-15% NaCl). All 25 strains were also screened for their ability to utilise xylose to produce xylitol. Meyerozyma caribbica was the most efficient xylitol producer, wherein three strains of this species (SRA4.6, SRA4.1, and SRA4.8) generated 18.61 to 21.56 g l -1 of xylitol, with 0.465-0.539 g g -1 yields. Through this study, we draw attention towards the tropical floral nectar as a potential niche for the isolation of diverse, osmotolerant, and xylitol-producing yeasts. Such osmotolerant yeasts have potential applications in food industries and biofuel production., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bloodstream infection survival is linked to timely administration of optimal antimicrobial therapy. Commercial multiplex polymerase chain reaction (PCR) assays, such as the BioFire Blood Culture Identification Panel (BCID) used for the rapid diagnosis of bloodstream infections, have significantly improved the turnaround time for optimisation of antimicrobial therapy. Reported concordance with culture-based methods and multiplex PCR analysis is high and only limited by (1) the range of targets available on the multiplex panel; and (2) the complexity of microorganisms present in the blood culture specimen. In this study, we evaluated the use of the BioFire Blood Culture Identification 2 panel (BCID2), including an expanded repertoire of targets for Gram-positive and Gram-negative bacteria, yeast and antimicrobial resistance genes compared to the BCID panel. The BCID2 panel identified microorganisms in 39/42 (92.9%) blood cultures where monomicrobial growth was detected; the three unidentified blood cultures contained organisms not included in the BCID2 panel. Polymicrobial blood culture analysis revealed a lower degree of concordance (28.6%); however, most disagreement was due to the culture-based identification of off-panel microorganisms of low clinical significance. Turnaround time, from blood culture collection to organism identification on the blood cultures correctly identified by BCID2, was 24.6 (±16.8) hours for the BCID2 panel versus 38.2 (±21.9) hours for conventional methods. Analysis of the theoretical impact of the BCID2 identification on clinical management found therapy would be altered in 45.1% (23/51) of patients. The BCID2 panel is anticipated to improve the diagnosis and antimicrobial management of patients with serious bloodstream infections., (Copyright © 2021 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)